Methods and compositions for detection of Zika viral infections

ABSTRACT

Applicant discloses herein kits for identifying the presence of Zika virus in a sample. In embodiments, these kits comprise reagents disclosed herein. Applicant further provides kits for use in detecting ZIKV in a sample, the kits comprising reagents disclosed herein. In embodiments, kits include primers directed to Zika virus (ZIKV) nucleic acid sequences, the primers capable of hybridizing to ZIKV nucleic acids and to copies of ZIKV nucleic acids (including to cDNA copies of ZIKV nucleic acids). Applicant discloses herein reagents for detecting Zika virus (ZIKV) in a sample, the reagents including one or more nucleic acid primers that are capable of hybridizing to a ZIKV nucleic acid (including to cDNA copies of ZIKV nucleic acids).

CROSS-REFERENCE

This application claims priority to U.S. Application Nos. 62/368,961filed Jul. 29, 2016, 62/368,995 filed Jul. 29, 2016, 62/369,006 filedJul. 29, 2016, 62/369,179 filed Jul. 31, 2016, and 62/369,009 filed Jul.29, 2016. All of the foregoing applications and patents are incorporatedherein by reference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 17, 2017, isnamed 3055_201_SL.txt and is 15,171 bytes in size.

BACKGROUND

A variety of methods for the amplification of nucleic acids are known.For example, polymerase chain reaction (“PCR”) (see, e.g. U.S. Pat. No.4,683,202) is a popular method for the amplification of nucleic acids.PCR methods are in vitro methods able to amplify a specificpolynucleotide sequence. PCR can be used to amplify specificpolynucleotide sequences, including genomic DNA, single-stranded cDNA,and mRNA among others. As described in U.S. Pat. No. 4,683,202, U.S.Pat. No. 4,683,195, and U.S. Pat. No. 4,800,159 (hereby incorporatedherein by reference), PCR typically comprises treating separatecomplementary strands of a target nucleic acid with two polynucleotideprimers to form complementary primer extension products on both strandsthat act as templates for synthesizing copies of the desired nucleicacid sequences. By repeating the separation and synthesis steps in anautomated system, essentially exponential duplication of the targetsequences can be achieved.

To successfully perform a PCR reaction, the reaction must be performedat multiple different temperatures. This requires hardware or othermechanisms for repeatedly changing the temperature of the PCR reaction.In embodiments where the target nucleic acid is RNA, reversetranscription PCR (rtPCR) may be used.

Zika virus (ZIKV) is a member of the Flavivirus genus of viruses (familyFlaviviridae). Other members of the genus include dengue virus (DENV),West Nile Virus (WNV), Japanese encephalitis virus (JEV), yellow fevervirus (YFV), and tick-borne encephalitic virus (TBEV). Flaviviruses havea single-strand, positive-sense RNA genome that serves both as a genomeand messenger RNA. The RNA genome is translated into a singlepolyprotein that is proteolytically cleaved into three structuralproteins (capsid, prM, and envelope) and non-structural proteins NS1 toNS5. The virion contains a nucleocapsid composed of the capsid protein(C) and the RNA genome, surrounded by an icosahedral shell comprisingboth the envelope (E) glycoprotein and membrane (M) protein or theprecursor membrane (prM) protein anchored in a lipid membrane.

Flaviviruses may be transmitted by the bite from an infected arthropod(e.g., mosquito or tick) and may cause widespread morbidity andmortality throughout the world. Most recently, the Zika virus has spreadrapidly across the Americas following its introduction into Brazil in2015. While Zika virus disease is usually mild with non-specificsymptoms, such as fever, rash, conjunctivitis, and muscle and jointpain, there have been more severe conditions linked to the Zika virus,such as congenital microcephaly in newborns and Guillain-Barré syndrome(GBS) in adults. Thus, it has become increasingly important to be ableto detect Zika virus infection in an individual.

Currently, Zika virus infection is diagnosed through detection of theviral RNA and virus isolation from blood samples, which can be timeconsuming. Diagnosis by serology can be difficult as the Zika virus cancross-react with other flaviviruses, such as DENV, WNV, and YFV. Thus,there is a need for better assays that are capable of specificallydetecting the Zika virus. Moreover, there is a need for a rapid andsimple test for detecting Zika virus infection at or near the point ofservice.

SUMMARY

In one embodiment, Applicant discloses herein reagents, methods, andkits for detecting Zika virus (ZIKV) in samples of bodily fluid.

Applicant discloses herein reagents for use in PCR methods for detectingZika virus (ZIKV) in samples of bodily fluid. In embodiments, providedherein is a method for detecting ZIKV in a sample of bodily fluid, themethod comprising: A) generating multiple complementary DNA (cDNA)copies of at least portions of ZIKV RNA, B) generating multiple copiesof said cDNA copies of ZIKV RNA by polymerase chain reaction (PCR)amplification. In embodiments of the methods disclosed herein,generating multiple complementary DNA (cDNA) copies of at least portionsof ZIKV RNA comprises using a reverse transcriptase to effect reversetranscription of said at least portions of ZIKV RNA to provide said cDNAcopies of at least portions of ZIKV RNA. In embodiments of the methodsfor detecting Zika virus (ZIKV) in samples of bodily fluid disclosedherein, the PCR methods comprise reverse transcription PCR (RT-PCR)methods. In embodiments, said cDNA copies comprise a polynucleotidetemplate for PCR amplification. In embodiments of the methods disclosedherein, generating multiple complementary DNA (cDNA) copies of at leastportions of ZIKV RNA comprises PCR amplification using a PCR reactionmixture that comprises a PCR amplification reaction first primer and aPCR amplification reaction second primer, wherein in the PCRamplification reaction mixture, the PCR amplification reaction firstprimer anneals to the polynucleotide template and the PCR second primeranneals to a polynucleotide which is complementary to the polynucleotidetemplate, and wherein in the PCR amplification reaction mixture,multiple copies of a PCR amplification reaction product are formed,wherein the PCR amplification reaction product is a double-strandednucleic acid molecule comprising a first strand and a second strand, andwherein a first strand of the PCR amplification reaction product is acopy of the polynucleotide template. In embodiments of the methods fordetecting Zika virus (ZIKV) in samples of bodily fluid disclosed herein,the PCR methods comprise real-time PCR methods. In embodiments of themethods for detecting Zika virus (ZIKV) in samples of bodily fluiddisclosed herein, the PCR methods comprise reverse transcriptionreal-time PCR methods.

In embodiments, a reagent for identifying the presence of ZIKV in asample comprises a nucleic acid that can serve as a positive control forPCR nucleic amplification assays for identifying the presence of ZIKV ina sample, and for detecting ZIKV in a sample. In embodiments, a reagentfor identifying the presence of ZIKV in a sample comprises a nucleicacid that can serve as a positive control for PCR nucleic amplificationassays for identifying the presence of ZIKV in a sample, and fordetecting ZIKV in a sample, and a buffer.

In embodiments, a reagent for identifying the presence of ZIKV in asample comprises a buffer and a variant of a nucleic acid sequence thatcomprises a nucleic acid sequence found in, or is complementary to anucleic acid sequence found in, or is homologous to a nucleic acidsequence found in, or complementary to a homologous nucleic acidsequence found in, ZIKV. In embodiments, such a nucleic acid sequencevariant has at least about 95% sequence identity to the nucleic acidsequence.

In embodiments, a reagent for identifying the presence of ZIKV comprisesa buffer selected from phosphate and tris(hydroxymethyl)aminomethane(TRIS). In embodiments, a reagent for identifying the presence of ZIKVcomprises a TRIS buffer.

In embodiments, Applicant provides herein reagents for identifying thepresence of ZIKV in a sample. In embodiments, a reagent for identifyingthe presence of ZIKV in a sample comprises a nucleic acid sequenceselected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and a buffer. In embodiments, thenucleic acid primer of a reagent as disclosed herein comprises a nucleicacid sequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2, andSEQ ID NO: 3.

In embodiments, a reagent for identifying the presence of ZIKV in asample comprises a buffer and a variant of a nucleic acid sequenceselected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, wherein the variant has at leastabout 95% sequence identity to the nucleic acid sequence. Inembodiments, the nucleic acid primer of a reagent as disclosed hereincomprises a variant of a nucleic acid sequence selected from the groupSEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein the variant has atleast about 95% sequence identity to the nucleic acid sequence.

In embodiments, a reagent for identifying the presence of ZIKV comprisesa buffer selected from phosphate and tris(hydroxymethyl)aminomethane(TRIS). In embodiments, a reagent for identifying the presence of ZIKVcomprises a TRIS buffer.

In embodiments of reagents for identifying the presence of ZIKV asdisclosed herein, the primer comprises a reporter molecule. Inembodiments wherein the primer comprises a reporter molecule, thereporter molecule may comprise a fluorescent moiety. In embodimentswherein the primer comprises a fluorescent moiety, the nucleic acidprimer may further comprise a quenching moiety effective to quenchfluorescence from the fluorescent moiety when the primer is nothybridized to a target nucleic acid sequence.

Applicant discloses herein kits for detecting Zika virus (ZIKV) insamples of bodily fluid. Kits for detecting Zika virus (ZIKV) in samplesinclude reagents as disclosed herein. Kits for detecting Zika virus(ZIKV) in samples include nucleic acids as disclosed herein. Kits fordetecting Zika virus (ZIKV) in samples include nucleic acid primers asdisclosed herein. Kits for detecting Zika virus (ZIKV) in samplesinclude nucleic acids comprising reporter molecules as disclosed herein.Kits for detecting Zika virus (ZIKV) in samples include nucleic acidprimers comprising reporter molecules as disclosed herein.

The assays and methods disclosed herein may be performed on a device, oron a system, for processing a sample. The assays and methods disclosedherein can be readily incorporated into and used in an automated assaydevice, and in an automated assay system. The assays and methodsdisclosed herein can be readily incorporated into and used in anautomated sample analysis device. For example, devices and systems asdisclosed herein may include a communication assembly for transmittingor receiving a protocol based on the analyte to be detected (e.g., ZIKV)or based on other analytes to be detected by the device or system.

Methods and compositions disclosed herein provide rapid assays whichrequire only small amounts of sample, such as only small amounts ofblood. Device and systems disclosed herein are configured to performsuch rapid assays which require only small amounts of sample, such asonly small amounts of blood. Accordingly, the methods, compositions,devices, and systems provide rapid tests, which require only smallbiological samples, and thus provide advantages over other methods,reagents, kits, assays, devices, and systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective image of a rRT-PCR Reagent kit and relatedarticles, as disclosed herein.

FIG. 2 is a plot of fluorescence (a measure of the numbers of cDNAcopies of the target nucleic acid sequence) versus time (as cycles)showing the results of rRT-PCR amplification of Zika virus.

FIG. 3 is a graph showing the copies resulting from rRT-PCRamplification of Zika virus.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. It may be notedthat, as used in the specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a material”may include mixtures of materials, reference to “a compound” may includemultiple compounds, and the like. References cited herein are herebyincorporated by reference in their entirety, except to the extent thatthey conflict with teachings explicitly set forth in this specification.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

As used herein, a “sample” may be, but is not limited to, blood,cerebrospinal fluid, bile, plasma, serum, saliva, sputum, mucus, andurine, or any portion thereof. The sample may be of any suitable size orvolume. In some embodiments of the assays and methods disclosed herein,measurements may be made using a small volume of the sample, or no morethan a small volume portion of the sample, where a small volumecomprises no more than about 5 mL; or comprises no more than about 3 mL;or comprises no more than about 2 mL; or comprises no more than about 1mL; or comprises no more than about 500 μL; or comprises no more thanabout 250 μL; or comprises no more than about 100 μL; or comprises nomore than about 75 μL; or comprises no more than about 50 μL; orcomprises no more than about 35 μL; or comprises no more than about 25μL; or comprises no more than about 20 μL; or comprises no more thanabout 15 μL; or comprises no more than about 10 μL; or comprises no morethan about 8 μL; or comprises no more than about 6 μL; or comprises nomore than about 5 μL; or comprises no more than about 4 μL; or comprisesno more than about 3 μL; or comprises no more than about 2 μL; orcomprises no more than about 1 μL; or comprises no more than about 0.8μL; or comprises no more than about 0.5 μL; or comprises no more thanabout 0.3 μL; or comprises no more than about 0.2 μL; or comprises nomore than about 0.1 μL; or comprises no more than about 0.05 μL; orcomprises no more than about 0.01 μL.

As used herein, the term “analyte” refers to a molecule of interest thatis detected or to be detected in an analytical procedure.

As used in the description herein and throughout the claims that follow,the meaning of “or” includes both the conjunctive and disjunctive unlessthe context expressly dictates otherwise. Thus, the term “or” includes“and/or” unless the context expressly dictates otherwise.

The term “moiety” as used herein refers to any particular composition ofmatter, e.g., a molecular fragment, an intact molecule, or a mixture ofmaterials.

As used herein, “nucleic acid” includes both DNA and RNA, including DNAand RNA containing non-standard nucleotides. A “nucleic acid” containsat least one polynucleotide (a “nucleic acid strand”). A “nucleic acid”may be single-stranded or double-stranded. This, the term “nucleic acid”refers to nucleotides and nucleosides which make up, for example,deoxyribonucleic acid (DNA) macromolecules and ribonucleic acid (RNA)macromolecules. Nucleic acids may be identified by the base attached tothe sugar (e.g., deoxyribose or ribose); as used herein, theabbreviations for these bases (shown in Table 1) are used to representnucleic acids in sequence listings identifying and describing theirstructures (either upper-case or lower-case may be used).

TABLE 1 Base (in Nucleic Acid) Letter Code Adenine A Thymine T Guanine GCytosine C Uracil U

RNA molecules found in nature consist of sequences of A, G, C, and U,while DNA molecules found in nature consist of A, G, C, and T; that is,where a RNA sequence that is complementary to a nucleic acid targetsequence includes U, a DNA sequence that is complementary to thatnucleic acid target sequence includes T in place of U.

As used herein, the term “polynucleotide” is used to refer to apolymeric chain containing two or more nucleotides. “Polynucleotides”include primers, oligonucleotides, nucleic acid strands, etc. Apolynucleotide may contain standard or non-standard nucleotides.Typically, a polynucleotide contains a 5′ phosphate at one terminus (“5′terminus”) and a 3′ hydroxyl group at the other terminus (“3′ terminus)of the chain. The most 5′ nucleotide of a polynucleotide may be referredto herein as the “5′ terminal nucleotide” of the polynucleotide. Themost 3′ nucleotide of a polynucleotide may be referred to herein as the“3′ terminal nucleotide” of the polynucleotide.

The term “downstream” as used herein in the context of a polynucleotidecontaining a 5′ terminal nucleotide and a 3′ terminal nucleotide refersto a position in the polynucleotide which is closer to the 3′ terminalnucleotide than a reference position in the polynucleotide. For example,in a primer having the sequence: 5′ ATAAGC 3′, the “G” is downstreamfrom the “T” and all of the “A”s.

The term “upstream” as used herein in the context of a polynucleotidecontaining a 5′ terminal nucleotide and a 3′ terminal nucleotide, refersto a position in the polynucleotide which is closer to the 5′ terminalnucleotide than a reference position in the polynucleotide. For example,in a primer having the sequence: 5′ TAGC 3′, the “T” is upstream fromthe “G”, the “C”, and the “A”.

As used herein, a nucleic acid molecule which is described as containingthe “sequence” of a template or other nucleic acid may also beconsidered to contain the template or other nucleic acid itself (e.g. amolecule which is described as containing the sequence of a template mayalso be described as containing the template), unless the contextclearly dictates otherwise.

As used herein “cDNA” refers to DNA molecules (“complementary DNA”)produced by reverse transcription of an RNA molecule. Such reversetranscription produces a DNA molecule having a nucleotide sequence thatis the same as the nucleotide sequence of that RNA molecule, with theexception that where the RNA molecule has a uracil moiety (U) the DNAmolecule has instead a thymine (T). A cDNA produced by reversetranscription of an RNA molecule is complementary to the complement ofthat RNA molecule.

As used herein, a “target” nucleic acid or molecule refers to a nucleicacid of interest. A target nucleic acid/molecule may be of any type,including single-stranded or double stranded DNA or RNA (e.g. mRNA). Insome instances, a target nucleic acid may be a nucleic acid which maydirectly function as a double-stranded nucleic acid template in a methodprovided herein (e.g. a double-stranded DNA molecule), or it may be anucleic acid which requires further processing or conversion to functionas a double-stranded nucleic acid template in a method provided herein(e.g. mRNA).

As used herein, “complementary” sequences refer to two nucleotidesequences which, when aligned anti-parallel to each other, containmultiple individual nucleotide bases which pair with each other. It isnot necessary for every nucleotide base in two sequences to pair witheach other for sequences to be considered “complementary”. Sequences maybe considered complementary, for example, if at least 30%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of thenucleotide bases in two sequences pair with each other. In addition,sequences may still be considered “complementary” when the total lengthsof the two sequences are significantly different from each other. Forexample, a primer of 15 nucleotides may be considered “complementary” toa longer polynucleotide containing hundreds of nucleotides if multipleindividual nucleotide bases of the primer pair with nucleotide bases inthe longer polynucleotide when the primer is aligned anti-parallel to aparticular region of the longer polynucleotide.

“Identical” or “identity,” as used herein in the context of two or morepolypeptide or polynucleotide sequences, can mean that the sequenceshave a specified percentage of residues that are the same over aspecified region. The percentage can be calculated by optimally aligningthe two sequences, comparing the two sequences over the specifiedregion, determining the number of positions at which the identicalresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the specified region, and multiplying the result by 100to yield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of the single sequence are included in thedenominator but not the numerator of the calculation.

“Homology” or “homologous” as used herein in the context of two or morepolypeptide or polynucleotide sequences, can mean that the sequenceshave a specified percentage of residues that are either i) the same, orii) conservative substitutions of the same residue, over a specifiedregion. Conservative substitutions include substitutions of one aminoacid by an amino acid of the same group, and include substitutions ofone amino acid by an amino acid as an exemplary or as a preferredsubstitution as known in the art. In determining homology of twosequences, identical residues and homologous residues are given equalweight. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which either identical orhomologous residues occur in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the specified region, and multiplying the resultby 100 to yield the percentage of sequence homology. In cases where thetwo sequences are of different lengths or the alignment produces one ormore staggered ends and the specified region of comparison includes onlya single sequence, the residues of the single sequence are included inthe denominator but not the numerator of the calculation.

As used herein, in the context of two or more polymeric molecules (e.g.nucleic acids, proteins), “corresponds to”, “corresponding to”, and thelike refers to polymeric molecules or portions thereof which have thesame or similar sequence of component elements (e.g. nucleotides, aminoacids). For example, if a first nucleic acid is described as containinga region which “corresponds to” the sequence of a second nucleic acid,the relevant region of the first nucleic acid has a nucleotide sequencewhich is the same or similar to the sequence of the second nucleic acid.

The term “primer” as used herein refers to a polynucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, i.e., in the presence of nucleotides and an inducing agent suchas DNA polymerase and at a suitable temperature and pH. The primer ispreferably single stranded for maximum efficiency in amplification, butmay alternatively be double stranded. If double stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. Preferably, the primer is apoly-deoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and use of the method. Forexample, for diagnostics applications, depending on the complexity ofthe target sequence, the polynucleotide primer typically contains about10-30 or more nucleotides, or about 15-25 or more nucleotides, althoughit may contain fewer nucleotides. For other applications, thepolynucleotide primer is typically shorter, e.g., 7-15 nucleotides. Suchshort primer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with template.

A primer may include a marker moiety, such as a fluorescent moiety, or aquencher moiety (e.g., for quenching fluorescence by fluorescence (orForster) resonance energy transfer FRET)), or combinations thereof.Fluorescein dyes (e.g., FAM™) are suitable fluorescent moieties (“dye”).Other suitable dyes include VIC®, ROX™, SYBR® Green, JOE, TAMRA™, andNED™ dyes, all of which are commercially available and may be linked tonucleic acid molecules. Dabcyl (4-((4-(dimethylamino)phenyl)azo)benzoicAcid) is an example of a suitable quencher which is commerciallyavailable and may be linked to nucleic acid molecules.

As used herein, when a first polynucleotide is described as “annealed”,“annealing” or the like to a second polynucleotide, the entirety of thefirst polynucleotide or any portion thereof may anneal to the secondpolynucleotide, and vice versa.

The “Tm” indicates the annealing temperature for a particular primerset; a primer set may have a different Tm than other primer sets, or mayhave the same Tm as another primer set. In many cases, Tm is typicallybetween about 45° C. to about 80° C., or between about 50° C. to about75° C.

As used herein, “reverse transcriptase” (RT) refers to an enzyme whichcan be used to produce a DNA molecule that is complementary to a RNAmolecule. The act of producing such a DNA molecule from an RNA templateis termed “reverse transcription”.

As used herein, polymerase chain reaction, abbreviated by the acronym“PCR”, refers to any of the nucleic acid amplification methods in whicha target nucleic acid (typically a double-stranded deoxyribonucleicacid) is exposed to a thermostable DNA polymerase during multiplethermal cycles, and in which multiple copies of the target nucleic acid(typically including copies of nucleic acid sequences disposed between afirst target region on one strand of a double-stranded target nucleicacid and a second target region on the complementary strand of adouble-stranded target nucleic acid) are produced, amplifying the targetnucleic acid. Thermal cycles typically include low temperature portions(typically at temperatures between about 40° C. and about 59° C., orbetween about 45° C. and about 55° C.), intermediate temperatureportions (typically at temperatures between about 60° C. and about 74°C.), and higher temperature portions (typically at temperatures betweenabout 75° C. and about 99° C., or between about 80° C. and about 95°C.). For example, some PCR reactions include a) incubation of a mixtureincluding target molecules and primers at high temperature (e.g., about90° C. to about 95° C.) to denature the target DNA; b) cooling themixture to an intermediate temperature (e.g., about 50° C. to about 60°C.) to allow annealing between the primers and target DNA; and c) in thepresence of DNA polymerase, generating extensions of the primers (e.g.,by action of the polymerase at, e.g. temperatures of about 65° C. toabout 75° C.); and repeating this cycle of steps a), b), and c). Stepsa), b), and c) together may be termed a “thermal cycle”.

Amplification occurs with each thermal cycle, and, following multiplecycles, significant amplification of the target nucleic acid moleculeproduces large numbers of DNA copies of the target sequence. PCRrequirements include a DNA polymerase (e.g., a thermostable DNApolymerase), deoxynucleotides, and appropriate buffer solutions. Where atarget nucleic acid is an RNA target, reverse transcriptase (RT) may beused to produce a DNA copy of the RNA, and PCR applied to the DNAcopies.

As used herein, reverse transcription PCR (RT-PCR) refers to methods foramplifying RNA targets, in which copy DNA molecules (cDNAs) are producedfrom RNA target polynucleotides by application of reverse transcriptase,and PCR is applied to the cDNA copies to amplify the cDNA copies fordetection and/or amplification of the target polynucleotide. RT-PCRrequirements include a reverse transcriptase, a DNA polymerase (e.g., athermostable DNA polymerase), deoxynucleotides (typically asdeoxynucleotide tri-phosphates (“dNTPs”) such as dATP, dTTP, dGTP, anddCTP), and appropriate buffer solutions.

As used herein, “real-time PCR” refers to PCR amplification methods inwhich the progress, or extent, of target amplification is monitoredduring the course of the assay (e.g., at each thermal cycle). Progressof the amplification reactions may be monitored, for example, detectingthe amount of fluorescence or absorbance of reporter molecules. Suitablereporter molecules include intercalating dyes (which are detectable whenbound to double-stranded DNA, or to the minor groove of DNA, such asethidium bromide and SYBR Green dye); fluorogenic probes, such asself-quenching dyes, or dye pairs (the pairs including a dye and aquencher) attached to primers (which fluoresce when the primer is boundto target, but do not produce significant fluorescence when nothybridized to target nucleic acid molecules); and other reportermolecules.

As used herein, “rRT-PCR” refers to reverse-transcription real-time PCR.rRT-PCR is real-time PCR applied to RNA targets, usingreverse-transcription PCR to amplify nucleic acids based on RNA targetmolecules, and monitoring the amplification using real-time PCR methods.As used herein, “rRT-PCR” refers to reverse-transcription real-time PCR.rRT-PCR is real-time PCR applied to RNA targets, usingreverse-transcription PCR to amplify nucleic acids based on RNA targetmolecules, and monitoring the amplification using real-time PCR methods.Reverse-transcription PCR methods provide the DNA substrate required forPCR by contacting a sample, under the appropriate conditions, with areverse transcriptase and producing cDNA copies of RNA molecules in thesample.

The terms “polypeptide” and “protein” may be used interchangeably torefer to molecules comprised of amino acids linked by peptide bonds.Individual amino acids may be termed “residues” of a polypeptide orprotein. The amino acid sequences of polypeptides disclosed herein maybe identified by SEQ ID NO: presented as a string of letters, where theletters have the following meanings:

TABLE 1B AminoAcid 3-Letter Code 1-Letter Code Alanine Ala A ArginineArg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acidGlu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

Amino acid sequence of polypeptides, including enzymes, may includevariants of a parent sequence with substitutions, insertions anddeletions as compared to the sequence of the parent polypeptide. Aminoacid variants of parent polypeptides may be suitable for the same orsimilar use as the parent polypeptide. For example, amino acid variantsof parent polypeptides having amino acid sequences that are 95% orgreater identical or similar to the amino acid sequence of the parentpolypeptide may be suitable for the same or similar use as the parentpolypeptide.

A composition may include a buffer. Buffers include, without limitation,phosphate, citrate, ammonium, acetate, carbonate,tris(hydroxymethyl)aminomethane (TRIS), (N-morpholino) propanesulfonicacid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO),2-(N-morpholino)ethanesulfonic acid (MES), N-(2-Acetamido)-iminodiaceticacid (ADA), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), cholamine chloride,N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES),acetamidoglycine, tricine(N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), glycinamide, andbicine (2-(Bis(2-hydroxyethyl)amino)acetic acid) buffers. Buffersinclude other organic acid buffers in addition to the phosphate,citrate, ammonium, acetate, and carbonate buffers explicitly mentionedherein.

In embodiments of the compositions disclosed herein, includingembodiments of the aqueous compositions and embodiments of the bufferedaqueous compositions, the composition may comprise albumin, gelatin,cytochrome C, an immunoglobulin, an amino acid, agar, glycerol, ethyleneglycol, a protease inhibitor, an antimicrobial agent, a metal chelatingagent, a monosaccharide, a disaccharide, a polysaccharide, a reducingagent, a chelating agent, or combinations thereof.

An article of manufacture may comprise a container; and a compositioncontained within the container, wherein the composition comprises anucleic acid molecule (such as, e.g., a primer directed to a targetrelated to ZIKA). An article of manufacture may comprise a container;and a composition contained within the container, wherein thecomposition comprises a nucleic acid molecule (such as, e.g., a primerdirected to a target related to ZIKA). An article of manufacture maycomprise a container; and a composition contained within the container,wherein the composition comprises a nucleic acid molecule (such as,e.g., a primer directed to a target related to ZIKA). The containers maybe formed from a variety of materials such as glass or plastic, and mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The article of manufacture may furthercomprise a label or package insert on or associated with the containerindicating that the composition may be used to detect the presence of anucleic acid molecule (such as, e.g., a primer directed to a targetrelated to ZIKA) in a sample.

Description and disclosure of examples of reagents, kits, assays,methods, kits, devices, and systems which may use, or be used with, thereagents, kits, methods, devices, and systems disclosed herein may befound, for example, in U.S. Pat. No. 8,088,593; U.S. Pat. No. 8,380,541;U.S. Pat. No. 8,435,738; U.S. Pat. No. 8,475,739; U.S. Pat. No.8,840,838; U.S. Pat. No. 9,250,229; U.S. Pub. No. 2014/0057255; U.S.Pub. No. 2013/0078624; WO 2013/052318; WO 2014/015199; U.S. patentapplication Ser. No. 14/183,503, filed Feb. 18, 2014; U.S. patentapplication Ser. No. 13/933,035, filed Jul. 1, 2013; U.S. patentapplication Ser. No. 13/769,820, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 14/183,503, filed Feb. 18, 2014; patent applicationSer. No. 14/214,850, filed Mar. 15, 2014; International PatentApplication PCT/US2014/030034, filed Mar. 15, 2014; International PatentApplication PCT/US2014/056151, filed Sep. 17, 2014; U.S. patentapplication Ser. No. 13/769,798, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 13/769,779, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 13/244,947 filed Sep. 26, 2011; PCT/US2012/57155,filed Sep. 25, 2012; U.S. application Ser. No. 13/244,946, filed Sep.26, 2011; U.S. patent application Ser. No. 13/244,949, filed Sep. 26,2011; and U.S. application Ser. No. 13/945,202, filed Jul. 18, 2013, thedisclosures of which patents and patent applications are all herebyincorporated by reference in their entireties.

In embodiments of such methods, the nucleic acid target moleculesamplified by the PCR amplification method comprise DNA. In embodimentsof such methods, the nucleic acid target molecules amplified by the PCRamplification method comprise RNA. In embodiments, the nucleic acid mayinclude uracil, and in embodiments may include dideoxyuracil (e.g., mayinclude dideoxyuracil in place of a thymine during amplification). Inembodiments of such methods, said second nucleic acid amplificationmethod comprises an isothermal nucleic amplification method.

In embodiments of the methods disclosed herein, primers used in theamplification methods are directed to a single target nucleic acidsequence, and its complement. In embodiments of the methods disclosedherein, primers used in the amplification methods are directed to aplurality of target nucleic acid sequences, and complements thereof.

In embodiments, the target molecule is a RNA target, and cDNA moleculesare generated from RNA target molecules by reverse transcription. ThesecDNA molecules provide substrate molecules for PCR amplification.

Optionally, the PCR amplification reaction first primer is at least 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or 60 and nomore than 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or100 nucleotides in length, and wherein when the PCR amplificationreaction first primer is annealed to the polynucleotide template, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the PCRamplification reaction first primer are mis-matched according toWatson-Crick base-pairing rules with corresponding nucleotides on thepolynucleotide template. Optionally, the PCR amplification reactionsecond primer is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 40, 50, or 60 and no more than 10, 11, 12, 13, 14, 15, 20, 25, 30,40, 50, 60, 70, 80, 90, or 100 nucleotides in length, and wherein whenthe PCR amplification reaction second primer is annealed to thepolynucleotide which is complementary to the polynucleotide template, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the PCRamplification reaction second primer are mis-matched according toWatson-Crick base-pairing rules with corresponding nucleotides on thepolynucleotide which is complementary to the polynucleotide template.

In embodiments, PCR methods for amplifying a polynucleotide targetmolecule include: A) generating multiple copies of a polynucleotidetemplate in a polymerase chain reaction (PCR) amplification reactionmixture, wherein the PCR amplification reaction mixture comprises afirst PCR amplification reaction primer and a second PCR amplificationreaction primer, wherein in the PCR amplification reaction mixture, thefirst PCR amplification reaction primer anneals to the polynucleotidetemplate and the second PCR amplification reaction primer anneals to apolynucleotide which is complementary to the polynucleotide template,and wherein in the PCR amplification reaction mixture, multiple copiesof a PCR amplification reaction product are formed, wherein the PCRamplification reaction product is a double-stranded nucleic acidmolecule comprising a first strand and a second strand, and wherein afirst strand of the PCR amplification reaction product is a copy of thepolynucleotide template.

Applicant discloses herein methods for detecting Zika virus (ZIKV) insamples of bodily fluid. In embodiments, provided herein is a method fordetecting ZIKV in a sample of bodily fluid, the method comprising: A)generating multiple complementary DNA (cDNA) copies of at least portionsof ZIKV RNA, B) generating multiple copies of said cDNA copies of ZIKVRNA by polymerase chain reaction (PCR) amplification. In embodiments ofthe methods disclosed herein, generating multiple complementary DNA(cDNA) copies of at least portions of ZIKV RNA comprises using a reversetranscriptase to effect reverse transcription of said at least portionsof ZIKV RNA to provide said cDNA copies of at least portions of ZIKVRNA. In embodiments of the methods for detecting Zika virus (ZIKV) insamples of bodily fluid disclosed herein, the PCR methods comprisereverse transcription PCR (RT-PCR) methods. In embodiments, said cDNAcopies comprise a polynucleotide template for PCR amplification. Inembodiments of the methods disclosed herein, generating multiplecomplementary DNA (cDNA) copies of at least portions of ZIKV RNAcomprises PCR amplification using a PCR reaction mixture that comprisesa PCR amplification reaction first primer and a PCR amplificationreaction second primer, wherein in the PCR amplification reactionmixture, the PCR amplification reaction first primer anneals to thepolynucleotide template and the PCR second primer anneals to apolynucleotide which is complementary to the polynucleotide template,and wherein in the PCR amplification reaction mixture, multiple copiesof a PCR amplification reaction product are formed, wherein the PCRamplification reaction product is a double-stranded nucleic acidmolecule comprising a first strand and a second strand, and wherein afirst strand of the PCR amplification reaction product is a copy of thepolynucleotide template. In embodiments of the methods for detectingZika virus (ZIKV) in samples of bodily fluid disclosed herein, the PCRmethods comprise real-time PCR methods. In embodiments of the methodsfor detecting Zika virus (ZIKV) in samples of bodily fluid disclosedherein, the PCR methods comprise reverse transcription real-time PCRmethods.

The assays and methods disclosed herein may be performed on a device, oron a system, for processing a sample. The assays and methods disclosedherein can be readily incorporated into and used in device forprocessing a sample, or a system for processing a sample, which may bean automated assay device, or may be an automated assay system. Such adevice, and such a system, may be useful for the practice of the methodsdisclosed herein. For example, a device may be useful for receiving asample. A device may be useful for preparing, or for processing asample. A device may be useful for performing an assay on a sample. Adevice may be useful for obtaining data from a sample. A device may beuseful for transmitting data obtained from a sample. A device may beuseful for disposing of a sample following processing or assaying of asample.

A device may be part of a system, a component of which may be anautomatic assay device. A device may be an automatic assay device. Anautomatic assay device may be configured to facilitate collection of asample, prepare a sample for a clinical test, or effect a chemicalreaction with one or more reagents or other chemical or physicalprocessing, as disclosed herein. An automatic assay device may beconfigured to obtain data from a sample. An automatic assay device maybe configured to transmit data obtained from a sample. An automaticassay device may be configured to analyze data from a sample. Anautomatic assay device may be configured to communicate with anotherdevice, or a laboratory, or an individual affiliated with a laboratory,to analyze data obtained from a sample.

A sample may be, for example, a blood sample (e.g., a sample obtainedfrom a fingerstick, or from venipuncture, or an arterial blood sample),a urine sample, a biopsy sample, a tissue slice, stool sample, or otherbiological sample; a water sample, a soil sample, a food sample, an airsample; or other sample. A blood sample may comprise, e.g., whole blood,plasma, or serum. An automatic assay device may receive a sample fromthe subject through a housing of the device. The sample collection mayoccur at a sample collection site, or elsewhere. The sample may beprovided to the device at a sample collection site.

Accordingly, Applicants disclose devices configured to measure ZIKV in asample of blood according to a method disclosed herein. Devicesconfigured to measure ZIKV in a sample of blood according to a methoddisclosed herein may be configured to determine ZIKV from a sample ofblood that comprises no more than about 1000 μL of blood, or no morethan about 500 μL of blood, no more than about 250 μL of blood, or nomore than about 150 μL of blood, or no more than about 100 μL of blood,or no more than about 50 μL of blood, or, in embodiments, wherein saidsample of blood comprises no more than about 25 μL of blood, or whereinsaid sample of blood comprises no more than about 10 μL, of blood, orwherein said sample of blood comprises less than about 10 μL of blood.Such devices may be configured to measure ZIKV in a sample of blood inless than about one hour, or, in embodiments, in less than about 40minutes, or in less than about 30 minutes.

In some embodiments, an automatic assay device may be configured toaccept or hold a cartridge. In some embodiments, an automatic assaydevice may comprise a cartridge. The cartridge may be removable from theautomatic assay device. In some embodiments, a sample may be provided tothe cartridge of the automatic assay device. Alternatively, a sample maybe provided to another portion of an automatic assay device. Thecartridge and/or device may comprise a sample collection unit that maybe configured to accept a sample.

A cartridge may include a sample, and may include reagents for use inprocessing or testing a sample, disposables for use in processing ortesting a sample, or other materials. Following placement of a cartridgeon, or insertion of a cartridge into, an automatic assay device, one ormore components of the cartridge may be brought into fluid communicationwith other components of the automatic assay device. For example, if asample is collected at a cartridge, the sample may be transferred toother portions of the automatic assay device. Similarly, if one or morereagents are provided on a cartridge, the reagents may be transferred toother portions of the automatic assay device, or other components of theautomatic assay device may be brought to the reagents. In someembodiments, the reagents or components of a cartridge may remainon-board the cartridge. In some embodiments, no fluidics are includedthat require tubing or that require maintenance (e.g., manual orautomated maintenance).

A sample or reagent may be transferred to a device, such as an automaticassay device. A sample or reagent may be transferred within a device.Such transfer of sample or reagent may be accomplished without providinga continuous fluid pathway from cartridge to device. Such transfer ofsample or reagent may be accomplished without providing a continuousfluid pathway within a device. In embodiments, such transfer of sampleor reagent may be accomplished by a sample handling system (e.g., apipette); for example, a sample, reagent, or aliquot thereof may beaspirated into an open-tipped transfer component, such as a pipette tip,which may be operably connected to a sample handling system whichtransfers the tip, with the sample, reagent, or aliquot thereofcontained within the tip, to a location on or within the automatic assaydevice. The sample, reagent, or aliquot thereof can be deposited at alocation on or within the automatic assay device. Sample and reagent, ormultiple reagents, may be mixed using a sample handling system in asimilar manner. One or more components of the cartridge may betransferred in an automated fashion to other portions of the automaticassay device, and vice versa.

A device, such as an automatic assay device, may have a fluid handlingsystem. The fluid may be a sample, a reagent, diluent, wash, dye, or anyother fluid that may be used by the device, and may include, but notlimited to, homogenous fluids, different liquids, emulsions,suspensions, and other fluids. A fluid handling system, includingwithout limitation a pipette, may also be used to transport vessels(with or without fluid contained therein) around the device. The fluidhandling system may dispense or aspirate a fluid. The sample may includeone or more particulate or solid matter floating within a fluid.

In embodiments, a fluid handling system may comprise a pipette, pipettetip, syringe, capillary, or other component. The fluid handling systemmay include one or more fluidically isolated or hydraulicallyindependent units. For example, the fluid handling system may includeone, two, or more pipette tips. The pipette tips may be configured toaccept and confine a fluid. The tips may be fluidically isolated from orhydraulically independent of one another. The fluid contained withineach tip may be fluidically isolated or hydraulically independent fromone fluids in other tips and from other fluids within the device. Thefluidically isolated or hydraulically independent units may be movablerelative to other portions of the device and/or one another. Thefluidically isolated or hydraulically independent units may beindividually movable. A fluid handling system may comprise one or morebase or support. A base or support may support one or more pipette orpipette units. A base or support may connect one or more pipettes of thefluid handling system to one another.

An automatic assay device may be configured to perform processing stepsor actions on a sample obtained from a subject. Sample processing mayinclude sample preparation, including, e.g., sample dilution, divisionof a sample into aliquots, extraction, contact with a reagent,filtration, separation, centrifugation, or other preparatory orprocessing action or step. An automatic assay device may be configuredto perform one or more sample preparation action or step on the sample.Optionally, a sample may be prepared for a chemical reaction and/orphysical processing step. A sample preparation action or step mayinclude one or more of the following: centrifugation, separation,filtration, dilution, enriching, purification, precipitation,incubation, pipetting, transport, chromatography, cell lysis, cytometry,pulverization, grinding, activation, ultrasonication, micro columnprocessing, processing with magnetic beads, processing withnanoparticles, or other sample preparation action or steps. For example,sample preparation may include one or more step to separate blood intoserum and/or particulate fractions, or to separate any other sample intovarious components. Sample preparation may include one or more step todilute and/or concentrate a sample, such as a blood sample, or otherbiological samples. Sample preparation may include adding ananti-coagulant or other ingredients to a sample. Sample preparation mayalso include purification of a sample. In embodiments, all sampleprocessing, preparation, or assay actions or steps are performed by asingle device. In embodiments, all sample processing, preparation, orassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample processing, preparation, or assayactions or steps are performed by a single device, and may be performedwithin a housing of a single device. In embodiments, many sampleprocessing, preparation, or assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

An automatic assay device may be configured to run one or more assay ona sample, and to obtain data from the sample. An assay may include oneor more physical or chemical treatments, and may include running one ormore chemical or physical reactions. An automatic assay device may beconfigured to perform one, two or more assays on a small sample ofbodily fluid. One or more chemical reaction may take place on a samplehaving a volume, as described elsewhere herein. For example one or morechemical reaction may take place in a pill having less than femtolitervolumes. In an instance, the sample collection unit is configured toreceive a volume of the bodily fluid sample equivalent to a single dropor less of blood or interstitial fluid. In embodiments, the volume of asample may be a small volume, where a small volume may be a volume thatis less than about 1000 μL, or less than about 500 μL, or less thanabout 250 μL, or less than about 150 μL, or less than about 100 μL, orless than about 75 μL, or less than about 50 μL, or less than about 40μL, or less than about 20 μL, or less than about 10 μL, or other smallvolume. In embodiments, all sample assay actions or steps are performedon a single sample. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all sample assayactions or steps are performed within a housing of a single device. Inembodiments, most sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, many sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

An automatic assay device may be configured to perform a plurality ofassays on a sample. In embodiments, an automatic assay device may beconfigured to perform a plurality of assays on a single sample. Inembodiments, an automatic assay device may be configured to perform aplurality of assays on a single sample, where the sample is a smallsample. For example, a small sample may have a sample volume that is asmall volume of less than about 1000 μL, or less than about 500 μL, orless than about 250 μL, or less than about 150 μL, or less than about100 μL, or less than about 75 μL, or less than about 50 μL, or less thanabout 40 μL, or less than about 20 μL, or less than about 10 μL, orother small volume. An automatic assay device may be capable ofperforming multiplexed assays on a single sample. A plurality of assaysmay be run simultaneously; may be run sequentially; or some assays maybe run simultaneously while others are run sequentially. One or morecontrol assays and/or calibrators (e.g., including a configuration witha control of a calibrator for the assay/tests) can also be incorporatedinto the device; control assays and assay on calibrators may beperformed simultaneously with assays performed on a sample, or may beperformed before or after assays performed on a sample, or anycombination thereof. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all of a plurality ofassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample assay actions or steps, of aplurality of assays, are performed by a single device, and may beperformed within a housing of a single device. In embodiments, manysample assay actions or steps, of a plurality of assays, are performedby a single device, and may be performed within a housing of a singledevice. In embodiments, sample processing, preparation, or assay actionsor steps may be performed by more than one device.

In embodiments, all of a plurality of assays may be performed in a shorttime period. In embodiments, such a short time period comprises lessthan about three hours, or less than about two hours, or less than aboutone hour, or less than about 40 minutes, or less than about 30 minutes,or less than about 25 minutes, or less than about 20 minutes, or lessthan about 15 minutes, or less than about 10 minutes, or less than about5 minutes, or less than about 4 minutes, or less than about 3 minutes,or less than about 2 minutes, or less than about 1 minute, or othershort time period.

An automatic assay device may perform nucleic acid assays, includingisothermal nucleic acid assays (e.g., assays for detecting and measuringnucleic acid targets in a sample, including DNA and RNA targets). Inembodiments, an automatic assay device may perform nucleic acid assaysas disclosed in U.S. patent application Ser. No. 14/183,503, filed Feb.18, 2014; U.S. patent application Ser. No. 14/214,850, filed Mar. 15,2014; International Patent Application PCT/US2014/030034, filed Mar. 15,2014; and in International Patent Application PCT/US2014/056151, filedSep. 17, 2014. An automatic assay device may perform antibody assays,including enzyme-linked immunosorbent assays (ELISA), and other assaysfor detecting and measuring the amounts of proteins (includingantibodies), peptides, and small molecules in samples. An automaticassay device may perform general chemistry assays, including electrolyteassays (e.g., assays for detecting and measuring the amounts ofelectrolytes such as sodium and potassium in a sample).

An automatic assay device may be configured to detect one or moresignals relating to the sample. An automatic assay device may beconfigured to identify one or more properties of the sample. Forinstance, the automatic assay device may be configured to detect thepresence or concentration of one analyte or a plurality of analytes or adisease condition in the sample (e.g., in or through a bodily fluid,secretion, tissue, or other sample). Alternatively, the automatic assaydevice may be configured to detect a signal or signals that may beanalyzed to detect the presence or concentration of one or more analytes(which may be indicative of a disease condition) or a disease conditionin the sample. The signals may be analyzed on board the device, or atanother location. Running a clinical test may or may not include anyanalysis or comparison of data collected.

A chemical reaction or other processing step may be performed, with orwithout the sample. Examples of steps, tests, or assays that may beprepared or run by the device may include, but are not limited toimmunoassay, nucleic acid assay, receptor-based assay, cytometric assay,colorimetric assay, enzymatic assay, electrophoretic assay,electrochemical assay, spectroscopic assay, chromatographic assay,microscopic assay, topographic assay, calorimetric assay, turbidmetricassay, agglutination assay, radioisotope assay, viscometric assay,coagulation assay, clotting time assay, protein synthesis assay,histological assay, culture assay, osmolarity assay, and/or other typesof assays, centrifugation, separation, filtration, dilution, enriching,purification, precipitation, pulverization, incubation, pipetting,transport, cell lysis, or other sample preparation action or steps, orcombinations thereof. Steps, tests, or assays that may be prepared orrun by the device may include imaging, including microscopy, cytometry,and other techniques preparing or utilizing images. Steps, tests, orassays that may be prepared or run by the device may further include anassessment of histology, morphology, kinematics, dynamics, and/or stateof a sample, which may include such assessment for cells.

A device may be capable of performing all on-board steps (e.g., steps oractions performed by a single device) in a short amount of time. Adevice may be capable of performing all on-board steps on a singlesample in a short amount of time. For example, from sample collectionfrom a subject to transmitting data and/or to analysis may take about 3hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 45minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes orless, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute orless. The amount of time from accepting a sample within the device totransmitting data and/or to analysis from the device regarding such asample may depend on the type or number of steps, tests, or assaysperformed on the sample. The amount of time from accepting a samplewithin the device to transmitting data and/or to analysis from thedevice regarding such a sample may take about 3 hours or less, 2 hoursor less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes orless, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3minutes or less, 2 minutes or less, or 1 minute or less.

A device may be configured to prepare a sample for disposal, or todispose of a sample, such as a biological sample, following processingor assaying of a sample.

In embodiments, an automatic assay device may be configured to transmitdata obtained from a sample. In embodiments, an automatic assay devicemay be configured to communicate over a network. An automatic assaydevice may include a communication module that may interface with thenetwork. An automatic assay device may be connected to the network via awired connection or wirelessly. The network may be a local area network(LAN) or a wide area network (WAN) such as the Internet. In someembodiments, the network may be a personal area network. The network mayinclude the cloud. The automatic assay device may be connected to thenetwork without requiring an intermediary device, or an intermediarydevice may be required to connect an automatic assay device to anetwork. An automatic assay device may communicate over a network withanother device, which may be any type of networked device, including butnot limited to a personal computer, server computer, or laptop computer;personal digital assistants (PDAs) such as a Windows CE device; phonessuch as cellular phones, smartphones (e.g., iPhone, Android, Blackberry,etc.), or location-aware portable phones (such as GPS); a roamingdevice, such as a network-connected roaming device; a wireless devicesuch as a wireless email device or other device capable of communicatingwireless with a computer network; or any other type of network devicethat may communicate possibly over a network and handle electronictransactions. Such communication may include providing data to a cloudcomputing infrastructure or any other type of data storageinfrastructure which may be accessed by other devices.

An automatic assay device may provide data regarding a sample to, e.g.,a health care professional, a health care professional location, such asa laboratory, or an affiliate thereof. One or more of a laboratory,health care professional, or subject may have a network device able toreceive or access data provided by the automatic assay device. Anautomatic assay device may be configured to provide data regarding asample to a database. An automatic assay device may be configured toprovide data regarding a sample to an electronic medical records system,to a laboratory information system, to a laboratory automation system,or other system or software. An automatic assay device may provide datain the form of a report.

A laboratory, device, or other entity or software may perform analysison data regarding a sample in real-time. A software system may performchemical analysis and/or pathological analysis, or these could bedistributed amongst combinations of lab, clinical, and specialty orexpert personnel. Analysis may include qualitative and/or quantitativeevaluation of a sample. Data analysis may include a subsequentqualitative and/or quantitative evaluation of a sample. Optionally, areport may be generated based on raw data, pre-processed data, oranalyzed data. Such a report may be prepared so as to maintainconfidentiality of the data obtained from the sample, the identity andother information regarding the subject from whom a sample was obtained,analysis of the data, and other confidential information. The reportand/or the data may be transmitted to a health care professional. Dataobtained by an automatic assay device, or analysis of such data, orreports, may be provided to a database, an electronic medical recordssystem, to a laboratory information system (LIS), to a laboratoryautomation system (LAS), or other system or software.

Reagents

Applicant discloses reagents herein, where the reagents are suitable foridentifying the presence of ZIKA virus in a sample, and are suitable fordetecting ZIKA virus in a sample. Methods for identifying the presenceof ZIKA virus in a sample, and for detecting ZIKA virus in a sample,include PCR nucleic acid amplification methods. In embodiments, the PCRnucleic acid amplification methods may be reverse transcription PCRmethods. In embodiments, the PCR nucleic acid amplification methods maybe real-time PCR methods. In embodiments, the PCR nucleic acidamplification methods may be real-time reverse transcription PCRmethods.

A reagent for identifying the presence of ZIKA virus in a sample, thereagent comprising a nucleic acid primer comprising a nucleic acidsequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and a buffer. Inembodiments, the nucleic acid primer comprises a nucleic acid sequenceselected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, andSEQ ID NO: 4.

A reagent for identifying the presence of ZIKA virus in a sample, thereagent comprising a variant of a nucleic acid primer comprising anucleic acid sequence selected from the group SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and abuffer, wherein the variant has at least about 95% sequence identity tothe nucleic acid sequence. In embodiments, the a variant of a nucleicacid primer comprises a variant of a nucleic acid sequence selected fromthe group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4,wherein the variant has at least about 95% sequence identity to thenucleic acid sequence.

The reagent for identifying the presence of a ZIKA virus in a sample,wherein the buffer is selected from phosphate and TRIS. In embodiments,the buffer is TRIS.

The reagent comprising a primer and a buffer, wherein the primercomprises a reporter molecule.

The reagent comprising a primer and a buffer, wherein the primercomprises a reporter molecule, and wherein the reporter moleculecomprises a fluorescent moiety, and the nucleic acid primer furthercomprises a quenching moiety effective to quench fluorescence from thefluorescent moiety when the primer is not hybridized to a target nucleicacid sequence.

Kits

A kit for identifying the presence of ZIKA virus in a sample, comprisinga comprises a reagent including a nucleic acid primer, the primercomprising a nucleic acid sequence selected from the group SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:6, and a buffer.

In embodiments, a kit may comprise a reagent including a nucleic acidprimer, the primer comprising a nucleic acid sequence selected from thegroup SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

In embodiments, a kit for identifying the presence of ZIKA virus in asample may include a reagent comprising a nucleic acid primer, theprimer comprising a variant of a nucleic acid sequence selected from thegroup SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO: 6, and a buffer, wherein the variant has at leastabout 95% sequence identity to the nucleic acid sequence.

In embodiments, a kit for identifying the presence of ZIKA virus in asample may include a reagent comprising a nucleic acid primer, thenucleic acid primer comprising a variant of a nucleic acid sequenceselected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, andSEQ ID NO: 4, and a buffer, wherein the variant has at least about 95%sequence identity to the nucleic acid sequence.

In embodiments, a kit for identifying the presence of ZIKA virus in asample may include a reagent including a nucleic acid primer, whereinthe nucleic acid primer comprises a reporter molecule.

In embodiments, a kit for identifying the presence of ZIKA virus in asample may include a reagent including a primer with a reportermolecule, wherein the reporter molecule comprises a fluorescent moiety,and the nucleic acid primer further comprises a quenching moietyeffective to quench fluorescence from the fluorescent moiety when theprimer is not hybridized to a target nucleic acid sequence.

In embodiments, a kit for identifying the presence of ZIKA virus in asample may include a reagent comprising a primer with a reportermolecule, wherein the reporter molecule comprises a fluorescent moiety,and the nucleic acid primer further comprises a quenching moietyeffective to quench fluorescence from the fluorescent moiety when theprimer is not hybridized to a target nucleic acid sequence.

In the following, abbreviations and acronyms have their standardmeanings. For example, “mM” means millimolar; “uL” and “μL” meanmicroliter; “PFU/uL” means plaque forming units per uL; “mg/ml” meansmilligram per milliliter; “BSA” means bovine serum albumin; “cp/uL”means copies per microliter; and “1E4” means 1×10⁴, the “E” indicatingthe exponent to the power of ten.

A kit for performing rRT-PCR for the detection of ZIKV contains primersand buffers. In embodiments, a kit includes the primers, probes, andpositive and sample processing controls for use in PCR assays. In suchan embodiment, the user needs to purchase commercially availableenzymes, negative control and buffer.

In a first embodiments, a kit for detection of Zika contains primers andbuffers and lacks enzymes, negative control, and buffer. Such a firstembodiment of a kit for performing rRT-PCR for the detection of ZIKV maycontain:

A) Sample processing control—MS2 bacteriophage supplied at 100× (200PFU/uL), used at 1× concentration (2 PFU/uL). Formulation buffer forMS2: 10 mM Tris pH 7.5, 1 mM MgCl₂, 100 mM NaCl, 0.1% gelatin, 2 mg/mlBSA. Volume=80 uL.

B) Zika virus positive control—Synthetic RNA target designed via invitro transcription of a g-block, supplied at 1E4 cp/uL, used at a finalconcentration of 4E3 cp/uL. Formulation buffer for positive control: 10mM Tris pH 8.0, 0.1 mM EDTA (IDTE, IDT Cat No. 11-05-01-09), 1 unit/μLRNase inhibitor (RI, NEB Cat No. M0314S). Volume=250 uL per tube, 4tubes provided.

C) Reagent A (Primer-probe mix)—comprises Zika primers (forward andreverse), MS2 primers (forward and reverse), Zika probe (FAMfluorophore, Dabcyl quencher), MS2 probe (SIMA-HEX fluorophore, Dabcylquencher). Formulation buffer for primers and probes: 10 mM Tris pH 8.0,0.1 mM EDTA (IDTE, IDT Cat No. 11-05-01-09) Volume=54 uL per tube, 4tubes provided.

Reagent A includes Zika primers (forward and reverse) and Zika probe:

RLX 4877 GACATGGCTTCGGACAG (SEQ ID NO: 1)

RLX4878 ATATTGAGTGTCTGATTGCTTG (SEQ ID NO: 2)

RLX4879 FAM TGCCCAACACAAGGTGAAGCC Dabcyl (SEQ ID NO: 3)

Reagent A includes MS2 probe (SIMA-HEX fluorophore, Dabcyl quencher):

RLX 4943 AACGAGTCATATGAATTTAGGC (SEQ ID NO: 4)

RLX 4944 GCAGCCCGATCTATTTTATTAT (SEQ ID NO: 5)

RLX 5156 HEX AGGGAACGGAGTGTTTACAGTTCC Dabcyl (SEQ ID NO: 6)

This kit works with venous serum, venous plasma, venous whole blood andcapillary whole blood matrices. This kit has been tested and found to besuitable for use in PCR assays. This kit is suitable for use on thefollowing RT-PCR platforms: Roche LightCycler® 480 Instrument II; BioRad CFX96 Touch™ Real Time PCR Detection System; and ABI 7500 Fast RealTime RT PCR.

A suitable thermo-cycling program used for PCR for such a kit isillustrated in the following table:

Temp Time Cycles 50° C. 30 min  1 95° C. 15 min  1 95° C. 15 sec 45 60°C.  1 min *

Fluorescence is measured after each of the 45 cycles, as indicated bythe asterisk (which indicates when fluorescence measurements are made).

In embodiments, an RT-PCR kit may include further components, including,for example, enzymes and additional controls (e.g., negative controls,sample processing controls, in addition to positive controls as providedin the kit embodiments discussed above).

In a second embodiment, an RT-PCR kit may comprise the following basiccomponents: Enzymes—reverse transcriptase (RT) and DNA polymerase;Reaction buffer—containing MgCl₂ and dNTPs besides salt, buffer;Primers—DNA oligonucleotides; Probes—DNA oligonucleotides with5′-fluorophore and 3′-quencher; and Controls—positive, negative, sampleprocessing.

For example, such a second embodiment of a kit for performing rRT-PCRfor the detection of ZIKV may contain:

Enzymes: reverse transcriptase; for example the following reversetranscriptase has the following amino acid sequence

(SEQ ID NO: 7): MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGPPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPRRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSKGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL.

A suitable DNA Taq polymerase for use in a second embodiment of a kit asdisclosed herein has the following amino acid sequence

(SEQ ID NO: 8): MGHHHHHHHHHHSSGHIEGRASSSGHENLYFQSMGMRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVERLAGHPFNLNSRDQLERVLEDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMEGVPREAVDPLMIRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLEPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLA VPLEVEVGIGEDWLSAKE.

A suitable formulation buffer for use in a second embodiment of a kit asdisclosed herein may be: 20 mM Tris pH 7.5, 300 mM KCl, 2 mM DTT, 0.1 mMEDTA, 50% Glycerol, 0.2% Triton X-100.

Thermal cycling parameters for PCR assays using reagents of a secondembodiment of a kit disclosed herein are presented in the followingtable. It will be understood that the number of cycles can be adjustedas desired; for example, the number of cycles may be decreased from 60to 45-50.

PCR cycle Temp Time Cycles 56° C. 10 mm  1x 95° C.  1 min  1x 95° C. 15sec 60x 64° C.  1 min*

The asterisk indicates that fluorescence may be measured after thisstep.

A reagent for use in a second embodiment of a kit having features asdisclosed herein may be termed a “mastermix” and may have a compositionas described in the following table:

# of # of reactions reactions Units [Stock] [Final] 1 60 water 7.01420.6 NEB 5X HF X 5 1 5 300 Phusion Buffer dNTP mM 10 0.2 0.5 30 BSA X100 3.5 0.88 52.5 MS2 Forward primer uM 150 1 0.17 10 MS2 Reverse primeruM 150 1 0.17 10 MS2 probe uM 25 0.3 0.3 18 Zika Forward primer uM 150 10.17 10 Zika Reverse primer uM 150 1 0.17 10 Zika probe uM 25 0.3 0.3 18RT RDP205 mg/mL 0.1 0.0005 0.13 7.5 His-tagged Taq mg/mL 0.25 0.002 0.212 (RDP282B) template 10 600 total 25 1500 Total MM 15 900 (no template)

The buffer indicated in the table above is Phusion® HF Buffer (NEB,catalog# B0518S) used after five-fold dilution.

A second embodiment of a kit having features as disclosed herein mayinclude dNTPs, which may be provided in solution, or may be provided indry (powdered) form.

A second embodiment of a kit having features as disclosed herein mayinclude BSA, for example, BSA for use following 100-fold dilution. BSAmay be provided in, and/or may be diluted in, a formulation buffer, suchas the following formulation buffer: 20 mM KPO₄ pH 7.0, 50 mM NaCl, 0.1mM EDTA, 5% Glycerol.

Kits as disclosed herein may be stored at −20° C. In embodiments, kitsas disclosed herein may be stored at other temperatures, such as, e.g.,at 0-4° C.; and at room temperature.

EXAMPLE

Thermal cycling protocols for polymerase chain reactions for nucleicacid amplification may include, for example: about 5 to 20 minutes at“low temperature” such as a temperature of between about 45° C. andabout 55° C., followed by about 1-15 minute at “high temperature” suchas a temperature of between about 80° C. to about 95° C., followed byabout 20 to 100 thermal cycles, where each thermal cycle consists ofabout 10 to 120 seconds at “high temperature” then about 1 to 15 minutesat “low temperature”. In some PCR methods, an “intermediate temperature”of between about 60° C. and about 74° C. may be applied, e.g., betweenapplications of “low temperature” and applications of “hightemperature”.

FIG. 1 shows components of a kit having features disclosed herein (e.g.,a Zika RT-PCR kit). The box with labeling on the outside is shown in aclosed configuration (on the right). The box with reagent vials is shownin an open configuration (central box in the figure) with the lid openand showing reagent vials in place in receptacles within the box. Thecontents of the reagent vials provide an integrated set of reagents toperform the assay. The kit may also include capillary sample collectiondevices and a shipment container. Shown in FIG. 1B are an open shipmentcontainer for transporting the samples stably, with each sample held ina Nanotainer™ (the left-most box in the figure), and three samplecollection devices (shown in the center foreground of the figure).

FIG. 2 provides results of a RT-PCR assays performed using reagents andusing automated sample analysis devices and systems as disclosed herein.

The primers used in the nucleic acid amplification Zika assay weredesigned from a consensus of a multi-sequence alignment of all Zikastrains deposited in GenBank. The gene target we selected is a 100-basepair region within the highly conserved polyprotein gene.

FIG. 3 presents inflection time measurements (average of fourreplicates) showing the average inflection time on the vertical axisplotted against the number of copies of the nucleic acid targetsequences in the sample that was analyzed.

An example of a suitable thermal cycling protocol useful for thepolymerase chain reaction portion of the rRT-PCR amplification is: 10minutes at 50° C., followed by 1 minute at 95° C., followed by 60thermal cycles; each thermal cycle consisted of 15 seconds at 95° C.then 1 minute at 64° C.

For further example, the following thermal cycling protocol was used forthe polymerase chain reaction portion of the rRT-PCR amplification: 30minutes at 50° C., followed by 15 minutes at 95° C., followed by 45thermal cycles; each thermal cycle consisted of 15 seconds at 95° C.then 1 minute at 60° C. Following the last thermal cycle (after thefinal 1 minute at 60° C.), fluorescence measurements may be taken.

Reagents suitable for use in PCR amplification methods include:

TABLE 2 RT-PCR formulation # of # of RT-PCR reactions reactions MasterMix Units [Stock] [Final] 1 60 water 7.62 457.00 NEB 5X HF X 5 1 5.00300.00 Phusion Buffer dNTP mM 10 0.2 0.50 30.00 BSA X 100 3.5 0.88 52.50Zika F RLX4877 uM 150 1 0.17 10.00 Zika R RLX4878 uM 150 1 0.17 10.00Zika probe RLX4879 uM 25 03 0.30 18.00 RT RDP205 mg/mL 0.1 0.0005 0.137.50 His-tagged Taq mg/mL 0.25 0.0025 0.25 15.00 (RDP282) template 10600 total 25 1500 Total MM (no 15.00 900.00 template)

5× Phusion buffer (HF & CG, (“high fidelity” and CG-rich) from NewEngland BioLabs (NEB) contains 7.5 mM MgCl₂ (1.5 mM at 1× (i.e., no)dilution).

Primer sequences used to identify ZIKV in a sample were:

TABLE 3 RLX 4877 GACATGGCTTCGGACAG SEQ ID NO: 1 RLX4878ATATTGAGTGTCTGATTGCTTG SEQ ID NO: 2 RLX4879 FAM TGCCCAACACAAGGTGAAGCCSEQ ID NO: 3 Dabcyl

Primer sequences used to identify MS2 in a sample were:

TABLE 4 RLX 4943 AACGAGTCATATGAATTTAGGC SEQ ID NO: 4 RLX 4944GCAGCCCGATCTATTTTATTAT SEQ ID NO: 5 RLX 5156 HEXAGGGAACGGAGTGTTTACAGTTCC SEQ ID NO: 6 Dabcyl

As shown, FIG. 2 shows the results of a RT-PCR amplification of Zikavirus, as relative fluorescence units (RFU) plotted against cycles(thermal cycles).

FIG. 2 is a plot of fluorescence (a measure of the numbers of cDNAcopies of the target nucleic acid sequence) versus time (as cycles)showing the results of rRT-PCR amplification of Zika virus.

The average Cq was calculated from four Cq measurements for differentnumbers of copies per reaction. These results are presented in thefollowing table (Table 5):

TABLE 5 T276A1 c/rxn in neg. In-house RT-PCR formulation sample prep Cq1Cq2 Cq3 Cq4 Cq(avg) Stdev 4,000 30.72 30.25 30.19 30.39 30.39 0.24 10036.31 35.95 34.97 35.74 35.74 0.57 50 36.29 36.32 37.14 36.10 36.46 0.4625 38.15 36.77 38.65 37.68 37.81 0.80 10 39.37 40.13 38.69 38.50 39.170.74 7.5 39.44 60.00 39.29 40.16 44.72 10.19 5 40.80 40.35 40.25 60.0045.35 9.77 0 60.00 60.00 60.00 60.00 60.00 0.00

FIG. 3 is a graph showing the average numbers of copies resulting fromrRT-PCR amplification of Zika virus as a function of copies perreaction.

As shown, FIG. 3 shows average Cq versus copies per reaction.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.

The publications discussed or cited herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.All publications and patent applications mentioned herein areincorporated herein by reference to disclose and describe the structuresand/or methods in connection with which the publications and/or patentapplications are cited.

The invention claimed is:
 1. A kit for identifying the presence of ZIKAvirus in a sample, comprising a nucleic acid primer comprising a nucleicacid sequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and a buffer. 2.The kit of claim 1, wherein the nucleic acid primer comprises a nucleicacid sequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2, andSEQ ID NO:
 3. 3. A kit for identifying the presence of ZIKA virus in asample, comprising a nucleic acid primer comprising a variant of anucleic acid sequence selected from the group SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and abuffer, wherein the variant has at least about 95% sequence identity tothe nucleic acid sequence.
 4. The kit of claim 3, wherein the nucleicacid primer comprises a nucleic acid primer comprising a variant of anucleic acid sequence selected from the group SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3, and a buffer, wherein the variant has at leastabout 95% sequence identity to the nucleic acid sequence.
 5. The kit ofclaim 1, wherein the buffer is selected from phosphate,tris(hydroxymethyl)aminomethane (TRIS), 3-(N-morpholino) propanesulfonicacid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO),2-(N-morpholino)ethanesulfonic acid (IVIES),N-(2-Acetamido)-iminodiacetic acid (ADA), andpiperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES).
 6. The kit of claim2, wherein the buffer is tris(hydroxymethyl)aminomethane (TRIS).
 7. Thekit of claim 1, further comprising a reporter molecule.
 8. The kit ofclaim 2, further comprising a reporter molecule.
 9. The kit of claim 3,further comprising a reporter molecule.
 10. The kit of claim 4, furthercomprising a reporter molecule.
 11. The kit of claim 1, wherein thenucleic acid primer comprises a reporter molecule.
 12. The kit of claim2, wherein the nucleic acid primer comprises a reporter molecule. 13.The kit of claim 3, wherein the nucleic acid primer comprises a reportermolecule.
 14. The kit of claim 4, wherein the nucleic acid primercomprises a reporter molecule.
 15. The kit of claim 11, wherein thereporter molecule comprises a fluorescent moiety, and the nucleic acidprimer further comprises a quenching moiety effective to quenchfluorescence from the fluorescent moiety when the primer is nothybridized to a target nucleic acid sequence.
 16. The kit of claim 12,wherein the reporter molecule comprises a fluorescent moiety, and thenucleic acid primer further comprises a quenching moiety effective toquench fluorescence from the fluorescent moiety when the primer is nothybridized to a target nucleic acid sequence.
 17. The kit of claim 13,wherein the reporter molecule comprises a fluorescent moiety, and thenucleic acid primer further comprises a quenching moiety effective toquench fluorescence from the fluorescent moiety when the primer is nothybridized to a target nucleic acid sequence.
 18. The kit of claim 14,wherein the reporter molecule comprises a fluorescent moiety, and thenucleic acid primer further comprises a quenching moiety effective toquench fluorescence from the fluorescent moiety when the primer is nothybridized to a target nucleic acid sequence.
 19. A kit for identifyingthe presence of ZIKA virus in a sample, comprising a nucleic acid primercomprising a variant of a nucleic acid sequence selected from the groupSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,and SEQ ID NO: 6, and a buffer, wherein the variant has at least about90% sequence identity to the nucleic acid sequence.
 20. The kit of claim19, further comprising a reporter molecule, wherein the reportermolecule comprises a fluorescent moiety, and the nucleic acid primerfurther comprises a quenching moiety effective to quench fluorescencefrom the fluorescent moiety when the primer is not hybridized to atarget nucleic acid sequence.